1. Field of the Invention
The present invention relates to a novel physiologically active peptide having immunoregulatory activities. More particularly, the present invention is concerned with a novel, substantially pure, physiologically active peptide having immunoregulatory activities, including the activity to inhibit multiplication of Toxoplasma (Toxoplasma gondii) inside cells, antibacterial activity, antiviral activity and antitumor activity, and a peptide composition comprising such a novel, substantially pure, physiologically active peptide. Also, the present invention is concerned with a pharmaceutical composition containing this substantially pure, physiologically active peptide as an active component, which composition is useful as an antiprotozoan, antibacterial, antiviral or antitumor agent.
In the present specification, amino acid residues are represented using abbreviations, as indicated below, approved by IUPAC-IUB Commission on Biochemical Nomenclature (CBN). With respect to amino acids and the like having isomers, those represented by the following abbreviations are of the L-configuration unless otherwise specified.
Gln: glutamine residue PA1 Asp: aspartic acid residue PA1 Pro: proline residue PA1 Tyr: tyrosine residue PA1 Val: valine residue PA1 Lys: lysine residue PA1 Glu: glutamic acid residue PA1 Ala: alanine residue PA1 Asn: asparagine residue PA1 Leu: leucine residue PA1 Phe: phenylalanine residue PA1 Gly: glycine residue PA1 His: histidine residue PA1 Ser: serine residue PA1 Thr: threonine residue PA1 Ile: isoleucine residue PA1 Trp: tryptophan residue PA1 Arg: arginine residue PA1 Met: methionine residue PA1 Cys: cysteine residue
2. Discussion of Related Art
Toxoplasma is a protozoan parasite which multiplies cheifly in mammalian endothelial cells, and Toxoplasma is feared because it is not only a pathogenic protozoan causing domestic animals to suffer from various diseases but also is a pathogen of human encephalomyelitis. Toxoplasma evades the killing action of a macrophage and, different from other microorganisms, repeats multiplication by binary fission due to endogeneous budding in a macrophage of a normal host.
In Toxoplasma-hyperimmune animals, it has been observed that the macrophages have the activity to kill and digest protozoa. It has been reported that the sera of such Toxoplasma-hyperimmune animals contain a humoral mediator (generally known as "cytokine") capable of inhibiting multiplication of Toxoplasma in the normal cells of the animals (see Sethi, K. K. et al., J. Immunol., vol. 131, pages 1151-1558, 1975).
It has also been reported that a cytokine capable of inhibiting multiplication of Toxoplasma in normal cells is present in the supernatant of a culture obtained by cultivating spleen cells excised from a Toxoplasma-hyperimmune animal in the presence of Toxoplasma lysate antigen (hereinafter referred to as "TLA") (see Shirahata, T., Shimizu, K., and Suzuki, N., Z. Parasitenkd, vol. 49, pages 11-23, 1976).
The above-mentioned cytokine is a glycoprotein having a molecular weight of from 30,000 to 40,000 which is believed to be a substance produced by sensitized T lymphocytes, and it is generally known as "Toxoplasma growth inhibitory factor" (hereinafter referred to simply as "Toxo-GIF") (see Shirahata, T., Shimizu, K., and Suzuki, N., Z. Parasitenkd, vol. 49, pages 11-23, 1976, and Nagasawa, H., et al., Immunobiol., vol. 156, pages 307-319, 1980). This Toxo-GIF inhibits multiplication of Toxoplasma not only in macrophages but also in other somatic cells. In this connection, it should be noted that Toxo-GIF only inhibits multiplication of Toxoplasma in the cells of the same animal species as the host and cannot inhibit multiplication of Toxoplasma in the cells of different animal species (see Nagasawa, H., et al, Immunobiol., vol. 156, pages 307-319, 1980 and Matsumoto, Y., et al., Zbl. Bakt. Hyg., vol. A 250, pages 383-391, 1981). Due to this species specificity, Toxo-GIF cannot be used for the prevention or curing of toxoplasmosis in human beings or various animals other than the host.
In these situations, the present inventor previously found that a low molecular weight substance obtained by hydrolysis of serum from a Toxoplasma-immune animal exhibits not only the species specificity-free activity to inhibit multiplication of Toxoplasma but also antimicroorganism activities against protozoa, bacteria and viruses as well as antitumor activities (see Japanese Patent Application Laid-Open Specification Nos. 57-142922/1982 and 57-144983/1982 and U.S. Pat. No. 4,482,543). The hydrolysate obtained from serum of a Toxoplasma-immune animal has been designated as "Obioactin" (see Suzuki, N., et al., Zbl. Bakt. Hyg. I. Abt. Orig., vol. A 256, pages 356-366, 1984). This Obioactin is a polypeptide substance having a molecular weight of not greater than 5000, and can inhibit multiplication of Toxoplasma not only in homologous cells but also in heterologous cells (see Suzuki, N., et al., Zbl. Bakt. Hyg. I. Abt. Orig., vol. A256, pages 367-380, 1984).
This native Obioactin is believed to be an aggregate or a mixture of a large number of various types of peptides. If it were possible to synthesize Obioactin by identifying active sites for each peptide, the usefulness of Obioactin would markedly increase. However, since Obioactin is believed to be an aggregate or a mixture of a large number of various types of peptides as mentioned above, it is extremely difficult to determine the amino acid sequence for each of the large number of various types of peptides constituting the aggregate or mixture by performing amino acid analysis of purified Obioactin fractions as obtained by chromatographic operations, thereby causing the synthesis of Obioactin to be unfeasible.
The present inventor also previously attempted amino acid analyses for a peptide contained in a fraction of Obioactin purified by reverse phase chromatography in which use was made of ODS-120T column (as described later). A plurality of amino acids were detected for each of the 1st position, the 2nd position, the 3rd position and so forth, as counted from the N-terminus, of the peptide contained in the fraction and, hence, it was impossible to directly determine an amino acid sequence for the peptide from the detected amino acids. However, in such attempts, it was surprisingly found that certain specific amino acid sequences exhibit desired activities (see U.S. Pat. No. 4,897,463). Accordingly, the specification of U.S. Pat. No. 4,897,463 discloses a substantially pure peptide comprising an amino acid sequence represented by a formula selected from the group consisting of formulae (7) to (10): EQU Gly-Glu-Glu-Glu-Glu-Glu (7), (SEQ ID NO. 1) EQU Glu-Glu-Glu-Glu-Glu (8), (SEQ ID NO. 2) EQU Asp-Asp-Asp-Asp-Asp (9), (SEQ ID NO. 3)
and EQU Ala-Glu-Glu-Glu-Glu-Glu (10), (SEQ ID NO. 4).
Among these peptides having the amino acid sequences represented by the above formulae (7) to (10), the peptide having the amino acid sequence represented by formula (7) exhibits the highest activity to inhibit multiplication of Toxoplasma inside cells. This peptide having the amino acid sequence represented by formula (7) has been designated by the present inventor as Obiopeptide-1 (hereinafter referred to simply as "Obio-1").